Prefabricated microparticle for performing a detection of an analyte

ABSTRACT

The present invention relates to a prefabricated microparticle for performing detection, preferably a digital detection and/or quantitation of an analyte. Furthermore, it also relates to a detection and/or quantitation of multiple analytes by prefabricated microparticles. It also relates to a collection of such prefabricated microparticles and to the use of such microparticle(s) and/or of such collection. Furthermore, the present invention also relates to a method of performing a detection and/or quantitation of an analyte in a sample wherein a microparticle or collection of microparticles are used. In one embodiment, in the collection of microparticles, individual microparticles are tailored for the detection of specific analytes and can be distinguished from each other by a specific label indicating the respective analyte for which the individual microparticle is specific.

The present invention relates to a prefabricated microparticle forperforming a detection and/or quantitation of an analyte. Furthermore italso relates to a detection and/or quantitation of multiple analytes byprefabricated microparticles. It also relates to a collection of suchprefabricated microparticles and to the use of such microparticle(s)and/or of such collection. Preferably the detection is a digitaldetection. Furthermore, the present invention also relates to a methodof performing a detection and/or quantitation of an analyte or ofmultiple analytes in a sample wherein a microparticle or a collection ofmicroparticles are used. In one embodiment, in the collection ofmicroparticles, individual microparticles are tailored for the detectionof specific analytes and can be distinguished from each other by aspecific label indicating the respective analyte for which theindividual microparticle is specific.

Numerous techniques and methods have been devised for the detection ofanalytes in a sample. The sensitive and quantitative detection of ananalyte is, in an ideal world, digital. To this end, the sample isdistributed to a number of reaction spaces, and in each reaction space,there is one analyte molecule at a maximum. In this manner, despite theoverall low amount of analyte, a high analyte concentration is reachedwith reference to the background, and thus the efficiency of thereaction is increased. The generated signal is concentrated to a smallconfined space and can thus be easily detected. As early as 1961, theactivity of individual enzyme molecules was measured in aqueous dropletsin oil (Rotman, 1961, PNAS, 47: pp. 1891-1991). This proved thefeasibility of the detection of activity of a single enzyme molecule andthus the possibility of performing digital assays. With the developmentand increased distribution of molecular amplification processes, theconcept of a “limiting dilution” found its way into the analytics ofnucleic acids (Sykes et al., 1992, Biotechniques, 13: pp. 444-449). Acompartmentalization was originally reached by dividing theanalyte/target molecule containing sample solution onto individualreaction spaces of a microtiter plate. Subsequently, a considerablyhigher number of reaction spaces was achieved by using capillaries andmicrostructured substrates (Kalinina et al. 1997, Nucleic AcidsResearch, 25: pp. 1999-2004). Likewise primer oligonucleotides whichwere immobilized to microparticles were used in combination withwater/oil immersions (Vogelstein et al. 1999, PNAS, 96: pp. 9236-9241).There are also formats available for performing digital immunoassays inwhich microstructured substrates are used which allow the generation ofsmall amounts of aqueous solution and which thus produce a plurality ofreaction spaces (Rissin et al. 2006, Nanolett. 6: pp. 520-523). Inessence, the methodology that is nowadays available for performingdetection of an analyte typically involve complex devices for thegeneration of micro reaction spaces or for performing the respectivedetection tests. Accordingly, it was an object of the present inventionto provide for a methodology for performing a detection, preferably adigital detection of an analyte in a sample which methodology is easy tohandle and which can be performed without extensive efforts on the partof the apparatuses used. It is also an object of the present inventionto provide for a methodology that is versatile and that can be tailoredtowards different analytes, yet is universally employable and can beeasily adapted to different analytes. It is furthermore an object toprovide for a detection method that allows for the enrichment ofanalytes from different volumes of liquid without having to adjust thefinal volume of the detection reaction.

In one aspect, the present invention relates to a method of performing adetection, preferably a digital detection, of an analyte in a sample,said method comprising the steps:

-   a) providing a prefabricated microparticle which has a surface and    includes a void volume for receiving an aqueous solution, wherein    said particle is dispersible in a non-aqueous medium and, upon    dispersion in a non-aqueous medium, provides for a defined reaction    space in such non-aqueous medium, in which defined reaction space a    chemical or biochemical reaction indicating the presence of an    analyte can be performed, and wherein said prefabricated    microparticle comprises a capture agent that, upon exposure of said    microparticle to a sample surrounding said prefabricated    microparticle and containing an analyte, selectively and    specifically binds the analyte to be detected and that, upon binding    of the analyte to the capture agent, forms a complex between said    capture agent and said analyte, wherein said capture agent binds the    analyte from a sample surrounding said prefabricated microparticle,    and wherein said prefabricated microparticle further comprises a    detection agent that is specific for the analyte or said complex    between said capture agent and said analyte, and that binds said    analyte or said complex between said capture agent and said analyte;-   b) exposing said prefabricated microparticle to an aqueous sample    suspected of containing an analyte to be detected, thus allowing the    capture agent to selectively and specifically bind the analyte to be    detected, if present;-   c) placing the prefabricated microparticle into a non-aqueous phase,    e.g. an oil phase and using the void volume of said prefabricated    microparticle as a defined reaction space in which a chemical or    biochemical reaction indicating the presence of an analyte, is    performed, by either-   d1) detecting the detection agent bound to said analyte or to said    complex between said capture agent and said analyte;    -   or-   d2) amplifying the analyte, if present, by way of an amplification    reaction, and detecting the thus amplified product by means of said    detection agent, wherein said analyte is a nucleic acid and said    amplification reaction is a nucleic acid amplification such as, for    example, PCR, TMA, NASBA, LAMP, 3SR, SDA, RCA, LCR, RPA, NEAR,    -   or-   d3) performing a signal amplification reaction, e.g. a nucleic acid    amplification if a nucleic acid is or forms part of said detection    agent, or e.g. an enzyme-based amplification of a signal, e.g. in    the form of a label, such as a dye or fluorophor, if an enzyme is or    forms part of said detection agent, and detecting the thus amplified    signal.

In one embodiment, said prefabricated microparticle is provided as aprefabricated microparticle which is dried, preferably freeze-dried.

In one embodiment, said prefabricated microparticle is not an in-situgenerated microparticle, preferably not a microparticle that is in-situgenerated at the site or in the reaction, at or during which analytedetection is to take place.

In one embodiment, the prefabricated microparticle is a porousmicroparticle.

In one embodiment, said prefabricated microparticle has an interstitialpore space that allows the microparticle to receive or take up a liquidsuch as an aqueous sample, and, if present, any solute therein, such asan analyte.

In one embodiment, the capture agent is predominantly located on thesurface of said prefabricated microparticle, such that the prefabricatedmicroparticle is capable of enriching and concentrating an analytelocated outside of the microparticle.

In one embodiment, said prefabricated microparticle is reconstituted inan aqueous solution, preferably either during step a) or step b), and,upon reconstitution, receives such aqueous solution in its void volume.

In one embodiment, said detection agent is included in saidprefabricated microparticle during a prefabrication process or isincluded in said aqueous solution the present invention and thus becomespart of the prefabricated microparticle upon reconstitution.

In one embodiment, said prefabricated microparticle is made of agel-forming agent, such gel-forming agent being preferably liquefiableupon the application of heat or light, or upon a change of pH, redoxpotential, ionic strength, temperature, magnetic field orelectromagnetic radiation, or upon exposure to an enzyme or, if thegel-forming agent itself comprises an enzyme, to a substrate of suchenzyme, or any combination of the foregoing.

In one embodiment, said gel-forming agent forms a matrix defining thesurface and the void volume of said microparticle. In one embodiment,said matrix is a porous matrix.

In one embodiment, said gel-forming agent is selected from the groupcomprising

-   a) synthetic polymers prepared from their corresponding monomers,    such as methylacrylate and acrylate, acrylamide and methacrylamide,    cyclic lactams, styrene-based monomers;-   b) silicone-based polymers, e.g. polydimethylsiloxanes and their    copolymers;-   c) naturally occurring polymers selected from polysaccharides, e.g.    agarose, chitin, chitosan, alginate, carrageenan, cellulose,    fucoidan, laminaran, gums selected from xanthan gum, arabic gum,    ghatti gum, guar gum, locust bean gum, tragacanth gum, karaya gum    and inulin; polypeptides, e.g. albumins, collagens, gelatins;    polynucleotides; and combinations thereof.

In one embodiment, said capture agent is selected from antibodies orantibody fragments, nucleic acids, including aptamers, Spiegelmers,non-antibody proteins capable of specifically binding an analyte oranalyte complex, such as receptors, receptor fragments, affinityproteins, e.g. streptavidin.

In one embodiment, said detection agent is selected from antibodies orantibody fragments, nucleic acids, including aptamers, Spiegelmers,non-antibody proteins, such as receptors, receptor fragments, affinityproteins, e.g. streptavidin, each of them optionally being labelled witha suitable reporter molecule, such as a dye, enzyme, chemical catalyst,or a mixture of reagents capable of starting a chemical reaction thatproduces an optically or otherwise detectable signal indicating thepresence of the analyte to be detected.

In one embodiment, said prefabricated microparticle is specificallylabelled.

In one embodiment, the method according to the present invention isperformed using a collection of prefabricated microparticles, saidprefabricated microparticles being as defined in any of the embodimentsof the present invention.

In one embodiment, in said collection of prefabricated microparticles,said prefabricated microparticles are different from each other in thatthey are specific for different analytes to be detected, wherein eachprefabricated microparticle is specifically labelled such that differentprefabricated microparticles and their corresponding detected analytescan be distinguished by the specific labels of the prefabricatedmicroparticles.

In one embodiment, said method involves the use of a prefabricatedmicroparticle or of a collection of prefabricated microparticles asdefined in the present invention, for performing a digital detection ofan analyte or a plurality of analytes in a sample or for enriching andconcentrating a plurality of analytes in a plurality of defined volumes,wherein, preferably, all of said defined volumes in said plurality ofdefined volumes are equal.

In one embodiment, after the step of exposing b), there is one orseveral washing steps.

In one embodiment, in step a), said prefabricated microparticles areprovided in dried faun, and, in step b), said prefabricatedmicroparticles are reconstituted in aqueous solution and then exposed toa sample suspected of containing an analyte to be detected, wherein,optionally after the step of reconstituting, there is one or severalwashing steps.

In one embodiment, in step b) the number of prefabricated microparticlesand the number of analyte molecules in the sample are maintained oradjusted, as necessary, such that the binding of a single analytemolecule per prefabricated microparticle follows a Poisson distribution,preferably such that, on average, there is no more than one analytemolecule bound per microparticle, thus allowing the detection of asingle analyte molecule per prefabricated microparticle.

In one embodiment, during step c), the prefabricated microparticle orthe collection of prefabricated microparticles is suspended in thenon-aqueous phase and/or is located on a solid substrate isolating eachprefabricated microparticle from other prefabricated microparticles, ifpresent, wherein, preferably, said solid substrate is a filter, a sieve,a substrate having a pattern of wells, recesses, grooves, channels,trenches, craters, holes, pillars or any combination of the foregoing.

In one embodiment, during or after step c), the gel-forming agent isliquefied, preferably through the application of heat or light, or by achange of pH, redox potential, ionic strength, temperature, magneticfield or electromagnetic radiation, or upon exposure to an enzyme or, ifthe gel-forming agent itself comprises an enzyme, to a substrate of suchenzyme, or any combination of the foregoing, resulting in an aqueousdroplet in a non-aqueous phase.

In one embodiment, said reaction space in which said chemical orbiochemical reaction indicating the presence of an analyte, isperformed, is defined by said void volume of said prefabricatedmicroparticle and is not substantially larger than said void volume ofsaid prefabricated microparticle.

In a further aspect, the present invention also relates to a method ofperforming a detection, preferably a digital detection, of an analyte ina sample, said method comprising the steps:

-   a) providing a prefabricated microparticle, which has a surface and    includes a void volume for receiving an aqueous solution, wherein    said particle is dispersible in an non-aqueous medium, and, upon    dispersion in a non-aqueous medium, provides for a defined reaction    space in such non-aqueous medium, in which defined reaction space a    chemical or biochemical reaction indicating the presence of an    analyte, can be performed, and wherein said prefabricated    microparticle preferably is a porous microparticle that, upon    exposure of said microparticle to a sample surrounding said    prefabricated microparticle and containing an analyte, binds and/or    immobilizes and/or receives the analyte from a sample surrounding    said prefabricated microparticle, in the pores of said    microparticle, by taking up a fraction of said sample in said pores,    and wherein said prefabricated microparticle further comprises a    detection agent that is specific for the analyte, and that binds    said analyte;-   b) exposing said prefabricated microparticle to an aqueous sample,    suspected of containing an analyte to be detected, thus allowing the    prefabricated microparticle to bind and/or immobilize and/or receive    the analyte to be detected, if present;-   c) placing the prefabricated microparticle into a non-aqueous phase,    e.g. an oil phase and using the void volume of said prefabricated    microparticle as a defined reaction space in which a chemical or    biochemical reaction indicating the presence of an analyte, is    performed, by either-   d1) detecting the detection agent bound to said analyte; or-   d2) amplifying the analyte, if present, by way of an amplification    reaction, and detecting the thus amplified product by means of said    detection agent, wherein said analyte is a nucleic acid and said    amplification reaction is a nucleic acid amplification such as, for    example, PCR, TMA, NASBA, LAMP, 3SR, SDA, RCA, LCR, RPA, NEAR, or-   d3) performing a signal amplification reaction, e.g. a nucleic acid    amplification, if a nucleic acid is or forms part of said detection    agent, or, e.g. an enzyme-based amplification of a signal, e.g. in    the form of a label, such a dye or a fluorophor, if an enzyme is or    forms part of said detection agent, and detecting the thus amplified    signal.

It should be noted that, in this aspect according to the presentinvention, the presence of a capture agent on the prefabricatedmicroparticle is not necessary, as long as the prefabricatedmicroparticle is capable of taking up liquid from the surroundings towhich it is exposed. For example, such microparticle may be a porousmicroparticle, thus allowing the uptake of liquid and of an analytepresent in said liquid in the porous microparticle, i.e. in the spaceprovided for by the pores of said microparticle. Thus, according to thisaspect of the present invention, the method even works if theprefabricated microparticle does not have a capture agent. Instead, anyanalyte present in a sample is received and taken up by a prefabricatedmicroparticle according to the present invention simply due to itscapability of receiving and taking up liquid therein. The presence of acapture agent increases the specificity and/or selectivity of theprefabricated microparticles for said analyte but is, however, notabsolutely required or essential for the prefabricated microparticlesaccording to the present invention to function. The presence of captureagents on the prefabricated microparticle may, however, in someembodiments increase the microparticle's capabilities of enrichingand/or concentrating an analyte from a sample.

In a further aspect, the present invention relates to a prefabricatedmicroparticle for performing a detection, preferably digital detectionof an analyte in a sample, wherein said microparticle has a surface andincludes a void volume for receiving an aqueous solution, wherein saidparticle is dispersible in a non-aqueous medium and, upon dispersion ina non-aqueous medium, is suitable to provide for a defined reactionspace in such non-aqueous medium, in which defined reaction space achemical or biochemical reaction indicating the presence of an analytecan be performed.

In one embodiment, the prefabricated microparticle according to thepresent invention is storable, preferably for a period of at least 2months, more preferably at least 6 months.

In one embodiment, the prefabricated microparticle according to thepresent invention is dried, preferably freeze-dried.

In one embodiment, the prefabricated microparticle according to thepresent is not an in-situ generated particle, preferably not a particlethat is in-situ generated at the site or in the reaction, at or duringwhich analyte detection is to take place.

In one embodiment, said prefabricated microparticle is a porousmicroparticle.

Preferably, said prefabricated microparticle has an interstitial porespace that allows the microparticle to receive or take up a liquid, suchas an aqueous sample, and, if present, any solute therein, such as ananalyte.

In one embodiment said prefabricated microparticle comprises a captureagent that, upon exposure of said microparticle to a sample surroundingsaid microparticle and containing an analyte, selectively andspecifically binds the analyte to be detected and that, upon binding ofthe analyte to the capture agent, forms a complex between said captureagent and said analyte, wherein said capture agent binds the analytefrom a sample surrounding said microparticle.

In one embodiment, said capture agent is predominantly located on thesurface of said microparticle, such that the microparticle is capable ofenriching and concentrating an analyte located outside of themicroparticle.

In one embodiment, the prefabricated microparticle according to thepresent invention further comprises a detection agent that is specificfor the analyte or said complex between said capture agent and saidanalyte, and that binds said analyte or said complex between saidcapture agent and said analyte.

In one embodiment, said prefabricated microparticle is reconstituted inan aqueous solution and, upon reconstitution, receives such aqueoussolution in its void volume.

In one embodiment, said detection agent is included in saidprefabricated microparticle during a prefabrication process or isincluded in said aqueous solution in which it is reconstituted and thusbecomes part of the microparticle upon reconstitution.

In one embodiment, said microparticle is made of a gel-forming agent,such gel-forming agent being preferably liquefiable upon the applicationof heat or light, or upon a change of pH, redox potential, ionicstrength, temperature, magnetic field or electromagnetic radiation, orupon exposure to an enzyme or, if the gel-forming agent itself comprisesan enzyme, to a substrate of such enzyme, or any combination of theforegoing.

In one embodiment, said gel-forming agent forms a matrix defining thesurface and the void volume of said microparticle. In one embodiment,said matrix is a porous matrix.

In one embodiment, said gel-forming agent is selected from the groupcomprising

a) synthetic polymers prepared from their corresponding monomers, suchas methylacrylate and acrylate, acrylamide and methacrylamide, cycliclactams, styrene-based monomers;b) silicone-based polymers, e.g. polydimethylsiloxanes and theircopolymers;c) naturally occurring polymers selected from polysaccharides, e.g.agarose, chitin, chitosan, alginate, carrageenan, cellulose, fucoidan,laminaran, gums selected from xanthan gum, arabic gum, ghatti gum, guargum, locust bean gum, tragacanth gum, karaya gum and inulin;polypeptides, e.g. albumins, collagens, gelatins; polynucleotides; andcombinations thereof.

In one embodiment, said capture agent is selected from antibodies orantibody fragments, nucleic acids, including aptamers, Spiegelmers,non-antibody proteins capable of specifically binding an analyte oranalyte complex, such as receptors, receptor fragments, affinityproteins, e.g. streptavidin.

In one embodiment, said detection agent is selected from antibodies orantibody fragments, nucleic acids, including aptamers, Spiegelmers,non-antibody proteins, such as receptors, receptor fragments, affinityproteins, e.g. streptavidin, each of them optionally being labelled witha suitable reporter molecule, such as a dye, enzyme, chemical catalyst,or a mixture of reagents capable of starting a chemical reaction thatproduces an optically or otherwise detectable signal indicating thepresence of the analyte to be detected.

In one embodiment, said microparticle is specifically labelled.

In a further aspect, the present invention also relates to a collectionof microparticles, said microparticles being as defined above.

In one embodiment, said microparticles are different from each other inthat they are specific for different analytes to be detected, whereineach microparticle is specifically labelled such that differentmicroparticles and their corresponding detected analytes can bedistinguished by the specific labels of the microparticles.

In a further aspect, the present invention relates to the use of amicroparticle according to the present invention or of a collection ofmicroparticles according to the present invention, for performing adigital detection of an analyte or a plurality of analytes in a sample.

In a further aspect, the present invention relates to the use of amicroparticle according to the present invention or of a collection ofmicroparticles according to the present invention for enriching andconcentrating an analyte in a defined volume, or for enriching andconcentrating a plurality of analytes in a plurality of defined volumes,wherein preferably, all of said defined volumes in said plurality ofdefined volumes are equal.

In a further aspect, the present invention relates to a method ofperforming a digital detection of an analyte in a sample, said methodcomprising the steps:

a) providing a collection of prefabricated microparticles according tothe present invention,b) exposing said collection to a sample suspected of containing ananalyte to be detected, thus allowing the capture agent to selectivelyand specifically bind the analyte to be detected, if present; wherein,optionally, after the step of reconstituting and/or the step ofexposing, there is one or several washing steps;c) placing the collection of microparticles into a non aqueous phase,e.g. an oil phase, andeitherd1) detecting the detection agent bound to said analyte or to saidcomplex between said capture agent and said analyte;ord2) amplifying the analyte, if present, by way of an amplificationreaction, and detecting the thus amplified product by means of saiddetection agent, wherein said analyte is a nucleic acid and saidamplification reaction is a nucleic acid amplification such as, forexample, PCR, TMA, NASBA, LAMP, 3SR, SDA, RCA, LCR, RPA, NEAR,ord3) performing a signal amplification reaction, e.g. a nucleic acidamplification if a nucleic acid is or forms part of said detectionagent, or e.g. an enzyme-based amplification of a signal, e.g. in theform of a label, such as a dye or fluorophor, if an enzyme is or formspart of said detection agent, and detecting the thus amplified signal.

In one embodiment, in step a), said prefabricated microparticles areprovided in dried form, and, in step b), said prefabricatedmicroparticles are reconstituted in aqueous solution and then exposed toa sample suspected of containing an analyte to be detected.

In one embodiment, in step b) the number of microparticles and thenumber of analyte molecules in the sample are maintained or adjusted, asnecessary, such that the binding of a single analyte molecule permicroparticle follows a Poisson distribution, preferably such that, onaverage, there is no more than one analyte molecule bound permicroparticle, thus allowing the detection of a single analyte moleculeper microparticle.

In one embodiment, during step c), the collection of microparticles issuspended in the non-aqueous phase and/or is located on a solidsubstrate isolating each micro particle from other microparticles,wherein, preferably, said solid substrate is a filter, a sieve, asubstrate having a pattern of wells, recesses, grooves, channels,trenches, craters, holes, pillars or any combination of the foregoing.

In one embodiment, during or after step c), the gel-forming agent isliquefied, preferably through the application of heat or light, or by achange of pH, redox potential, ionic strength, temperature, magneticfield or electromagnetic radiation, or upon exposure to an enzyme or, ifthe gel-forming agent itself comprises an enzyme, to a substrate of suchenzyme, or any combination of the foregoing, resulting in an aqueousdroplet in a non-aqueous phase.

In a further aspect, the present invention also relates to a method formaking a prefabricated microparticle in accordance with the presentinvention, wherein said method comprises

-   a) providing, in any order, an aqueous phase including a gel-forming    agent, and separate from said aqueous phase, an oil phase,-   b) forming aqueous droplets of the aqueous phase including the    gel-forming agent within the oil phase, preferably by generating a    stream of the oil phase and by dosing in defined volumes of aqueous    phase into said flowing stream of said oil phase,-   c) collecting the thus generated aqueous droplets within said oil    phase and subsequently separating said aqueous droplets from said    oil phase by mechanical separation, such as centrifugation, sieving    or filtering.

The present inventors have devised a methodology wherein, in contrast tothe prior art, prefabricated microparticles are used in and for a methodof performing a detection, in which said prefabricated microparticlesthemselves define and limit the reaction space in which said detectiontakes place. In other words, in accordance with embodiments of thepresent invention, the prefabricated microparticle(s) is (are) providedand is (are) used as a detection compartment. Thus, the dimensions ofthe microparticle(s) of the present invention define and limit thedimensions of said compartment in which a detection takes place. Incontrast thereto, according to the prior art, in many instances, solidbeads are used in which capture agents are attached to the surfacethereof, and these beads themselves act to catch, immobilize or capturean analyte, but for a subsequent detection reaction these beads orparticles are incorporated in droplets or reaction spaces which areconsiderably larger than said beads or particles. In contrast thereto,in accordance with embodiments of the present invention, a prefabricatedmicroparticle itself defines and limits the dimensions of the reactionspace in which a detection reaction takes place. Thus, the dimensions ofthe prefabricated microparticle in accordance with the present inventionare the dimensions of the reaction space in which detection of ananalyte takes place. Without wishing to be bound by any theory, thepresent inventors therefore consider their methodology as the first andonly microparticle-mediated compartmentalization for a detectionreaction. This distinguishes the present invention from all of the priorart methodologies described above. Thus, in accordance with embodimentsof the present invention, the prefabricated microparticle, after havingbeen exposed to an aqueous sample suspected of containing an analyte tobe detected, is subsequently not incorporated in a larger aqueousdroplet surrounded by an oil phase. Likewise, in accordance withembodiments of the present invention, the prefabricated microparticleafter having been exposed to an aqueous sample suspected of containingan analyte to be detected, is also not placed into a larger compartment,such as a well or microreactor where it is combined with otherconstituents of an aqueous solution, including a volume of water, toform an aqueous droplet that is considerably larger than themicroparticle itself. Instead, in accordance with embodiments of thepresent invention, the prefabricated microparticle itself defines andlimits the reaction space/reaction compartment in which detection of ananalyte takes place.

Hence, in accordance with embodiments of the present invention, theprefabricated microparticle(s) of the present invention provides avolume-defining scaffold which is or becomes filled with a aqueoussolution to such an extent that the total void volume of saidprefabricated microparticle is filled, or only a fraction thereof. Inother words, in accordance with embodiments of the present invention,the total volume of the reaction space in which the detection of ananalyte takes place, is limited at the upper end by the maximum volumeprovided for by the prefabricated microparticle and is factually limitedby the total volume of liquid or liquid sample taken up by saidprefabricated microparticle. Upon incorporation of said liquid-filledprefabricated microparticle in a non-aqueous phase, the liquid-filledvolume of said prefabricated microparticle thus represents and acts as areaction space in which a detection reaction of the analyte takes place.

Such a compartmentalization of a reaction space by way of aprefabricated microparticle which itself acts as a volume scaffold toprovide for such reaction space, has not been described before.

Micro-droplet generating devices for performing such methods and forgenerating aqueous droplets do exist and may be readily adapted to thepresent invention. For example, devices that are useful for the presentinvention are dosing devices from Dolomite Microfluidics, UK. Suchdevices are also further described in WO 2002/068104 and WO 2005/089921.The devices described therein can be adapted to generate aqueousmicrodroplets within an oil phase, in accordance with embodiments of thepresent invention. In a further embodiment, the separated aqueousdroplets generated by the above method, in particular after step c) canbe washed using an aqueous solution or water. Furthermore, subsequently,they can be dried, e.g. freeze-dried. In accordance with the presentinvention, the aqueous droplets thus produced are prefabricatedmicroparticles in accordance with the present invention. In oneembodiment, a gel forming agent may be used for forming the aqueousdroplets/prefabricated microparticles, and such gel-forming agent is asdefined further above. In one embodiment, the aqueous droplets includingthe gel-forming agent are dried, preferable freeze-dried. Alternatively,any other suitable means of stripping off the solvent may be employed.Once the solvent has been removed and the aqueous droplet/producedmicroparticle has been dried, it may be stored as a powder. The presentinventors have surprisingly found that by providing prefabricatedmicroparticles in accordance with the present invention, it is possibleto provide miniaturized and defined reaction spaces that may be used ina very versatile manner for detection reactions, for example forperforming a digital detection of an analyte in a sample. Theprefabricated microparticles, in accordance with embodiments of thepresent invention, may be tailor made by choosing an appropriate captureagent that is comprised by the prefabricated microparticle and that,upon exposure of the microparticle to a sample that surrounds themicroparticle and that contains an analyte, selectively and specificallybinds the analyte to be detected. Because of their defined size, themicroparticles take up a defined volume of liquid and thus allow any(detection) reaction to take place in a defined volume of liquid.Effectively, the particles provide an efficient and easy means toportion a sample suspected of containing an analyte to be detected intowell and clearly defined small volumes. The microparticles in accordancewith embodiments of the present invention also provide an easy means toselectively enrich and/or concentrate the analyte to be detectedselectively on the surface of the microparticle. If desired, theyfurthermore allow the achievement of a uniform and standardizedconcentration of analyte stemming from different samples havingdifferent volumes and different starting concentrations of analyte.Different microparticles may be specific for different analytes to bedetected by choice of appropriate capture agent(s). Moreover, dependingon their respective specificity for an analyte to be detected, differentmicroparticles, in accordance with embodiments of the present invention,may be specifically labeled such that different microparticles and theircorresponding detected analytes can be distinguished by the specificlabels of the microparticles. Such specific labelling and thedistinction that can be achieved thereby is herein also sometimesreferred to as “encoding”. An “encoded” microparticle is a microparticlethat has been made specific, in terms of its binding capabilities, for aparticular analyte and that has also been marked or labelledspecifically accordingly. According to one embodiment, the prefabricatedmicroparticles are made of a gel-forming agent. In one embodiment, thegel-forming agent may exist in two different states, one state being asolid state or semi-solid state, the other state being a liquid state.In one embodiment, in the solid state or semi-solid state, thegel-forming agent is present in the form of a gel which forms a matrix,and, with such gel, the microparticles may, for example, be in the formof a suspension wherein the microparticles include a volume of anaqueous solution and are dispersed in a non-aqueous medium, such as anoil medium. Effectively, in this state, the microparticles representaqueous droplets that are reinforced by a matrix formed by thegel-forming agent/gel. As outlined further above, such matrix definesthe surface and the void volume of the microparticle. In a furtherembodiment, the gel-forming agent may be transferred from thesolid/semi-solid state into a liquid state upon the application of anappropriate stimulus. Such stimulus may be for example the applicationof heat or light or it may involve a change of pH, redox potential,ionic strength, temperature, magnetic field or electromagneticradiation. Alternatively, such external stimulus may also be theexposure to an enzyme (which, for example, may digest the matrix formedby the gel-forming agent), or, if the gel-forming agent itself comprisesan enzyme, such stimulus may be exposure to a substrate of such enzyme.Also combinations of any of the foregoing stimuli are envisaged. Oncethe gel-forming agent has been transferred from the solid/semi-solidstate to a liquid state, there will result an aqueous droplet in anon-aqueous medium (e. g. oil). As long as the gel-forming agent is inthe solid/semi-solid state, the microparticles are in the form of asuspension of such solid/semi-solid particles in a non-aqueous phase.Once the gel-forming agent has been liquefied, the microparticles are inthe form of an emulsion of an aqueous phase in a non-aqueous phase.

The prefabricated microparticles in accordance with embodiments of thepresent invention are storable, in particular in a dry state or driedstate, preferably for a period of at least two months, more preferablyfor a period of at least six months. In one embodiment, they arestorable for a period of at least one year. In one embodiment, theprefabricated microparticle according to the present invention maycomprise one or several stabilizing agents helping to preserve themicroparticle. Examples of such stabilizing agents are cyclodextrins(e.g. Cavasol®), trehalose, sucrose, lactose, mannose, glucose,galactose, mannitol, myoinositol, poly(alkylene oxides), in particularpoly(ethylene glycols) and their derivatives. The term “prefabricated”,as used herein, is meant to differentiate themicroparticle/microparticles according to the present invention fromother microparticles from the prior art which may possibly be used fordetection purposes, in that the prefabricated microparticle(s) inaccordance with the present invention is (are) not a particle(particles) that is (are) generated at the time and/or place of its(their) intended use. Hence, a prefabricated microparticle according tothe present invention is not an in-situ generated particle, i. e. it isnot a particle that is generated in the course of the reaction, e. g.the analytic assay, in which it is intended to be used. In particular,it is not generated at the site or time or reaction at, in or duringwhich an analyte detection is to take place. The term “in-situgenerated”, as used herein, is meant to refer to a substance or particlethat is generated from one or more precursors at the place and/or timeof intended use of such substance or particle. Moreover, a prefabricatedmicroparticle, in accordance with the present invention, is not aparticle that, at the time of its being generated, is made to encompassor include or incorporate or engulf a sample containing an analyte.Rather, a prefabricated microparticle in accordance with the presentinvention is generated first and, optionally, further processed, e. g.washed, dried, reconstituted etc.; and only after its generation, aprefabricated microparticle according to the present invention then isexposed to a sample containing an analyte or suspected of containing ananalyte.

The term “microparticle”, as used herein, is meant to refer to aparticle the average dimensions of which are in the micrometer range. Inone embodiment, the microparticles in accordance with the presentinvention have an average size or average dimension or average diameterof approximately 5 μm-200 μm, preferably 5 μm-150 μm, more preferably 10μm-100 μm. In one embodiment, the microparticles in accordance with thepresent invention are spherical or oval or ellipsoidal, preferablyspherical, and the above-mentioned dimensions refer to the averagediameter of such spherical, oval or ellipsoidal microparticle. In oneembodiment, the microparticles have the shape of a (spherical) droplet.In another embodiment, a microparticle in accordance with the presentinvention is a spherical body or a quasi-spherical body, i. e. havingthe shape of a sphere (or nearly approaching it), such sphere having anaverage diameter of the aforementioned dimensions. In one embodiment, amicroparticle in accordance with the present invention is porous. In afurther embodiment, a microparticle in accordance with the presentinvention, in particular a porous microparticle, has a surface that isavailable for accommodating a capture agent, in that the capture agentis predominantly located on the surface of the microparticle. In oneembodiment, the surface of a porous spherical microparticle inaccordance with the present invention having a defined diameter, isx-times the surface of a non-porous microparticle having the samediameter, with x being selected from at least 2, at least 5, at least10, at least 50, at least 100 or at least 500. In such a porousspherical microparticle in accordance with the present invention, thedensity of capture agent per microparticle is greatly enhanced andallows for a particularly efficient concentration of analyte to bedetected at the surface of the microparticle. This is because thedensity of capture agent on the surface of the microparticle is alsoparticularly high.

The microparticle(s) in accordance with embodiments of the presentinvention are also characterized by the fact that, when being generatedor when in use, they do not incorporate or include or encompass abiological cell. Likewise, when being generated or when in use, theyalso do not include or incorporate or encompass an analyte in theirinterior. Rather, any analyte that is to be detected by means of theprefabricated microparticle according to the present invention isselectively and specifically bound by the prefabricated microparticle atits surface, with the analyte being located in or stemming from a samplesurrounding the prefabricated microparticle. Hence, in one embodiment,the microparticle according to the present invention comprises a captureagent that, upon exposure of the microparticle to a sample surroundingthe microparticle and containing an analyte, selectively andspecifically binds the analyte to be detected, wherein the capture agentbinds the analyte from a sample surrounding the microparticle (and doesnot bind an analyte from a sample that is located within the particle).In one embodiment, the microparticle comprises a capture agent that ispredominantly located on the surface of the microparticle, andconsequently, the microparticle is thus capable of enriching andconcentrating an analyte located outside of the microparticle. The term“predominantly located”, when used in conjunction with a capture agentbeing located on the surface of a microparticle, is meant to refer to ascenario wherein the majority of such capture agent molecules arelocated on the surface of the microparticle rather than in its interior.As used herein, the term “surface” is meant to refer to the part of amicroparticle that is accessible from the outside of the microparticle.Likewise, as used herein, the term “interior of a microparticle” ismeant to refer to the part of a microparticle that is not accessible tothe outside of the microparticle. In one embodiment according to thepresent invention, the microparticle according to the present inventiondoes not encapsulate or encompass an analyte or a biological cell or amicroorganism, such as a bacterium, and hence does not contain suchanalyte in its interior.

In accordance with embodiments of the invention, a microparticle willhave an inherently (limited) capability of comprising or accommodating acapture agent. Hence, in one embodiment of a collection ofmicroparticles, preferably, the individual microparticles will haveapproximately the same density of capture agents, i. e. the same numberof capture agents per unit surface of microparticle. This will allow themicroparticles to enrich and concentrate an analyte to approximately thesame concentration, even when different samples having differentconcentrations of analyte, are used. Thus, the prefabricatedmicroparticles according to embodiments of the present invention alsoallow the generation of multiple identical reaction spaces/volumes,preferably with a uniform concentration of analyte at the surface of themicroparticles, after the microparticles have been exposed to a samplecontaining an analyte.

As used herein, the term “digital detection”, when used in conjunctionwith microparticles according to the present invention, is meant torefer to a scenario wherein either the ratio of the number ofmicroparticles to the number of analyte molecules is adjusted such thatthere is maximally a single analyte molecule bound per microparticle andthe binding of a single analyte molecule per microparticle follows aPoisson distribution. Alternatively or additionally the term “digitaldetection” when used in conjunction with microparticles according to thepresent invention, is meant to refer to a scenario wherein a sample isportioned by means of a collection of microparticles according to thepresent invention such that each microparticle provides the samereaction volume and reaction conditions and, preferably also containsapproximately or exactly the same number of analyte molecules. In thelatter scenario, the microparticles thus serve to create a plurality oflike reaction spaces (e. g. detection spaces) for each analyte type tobe detected, in which reaction spaces preferably the individualconcentrations of analyte are the same (or nearly identical within theerror margin) amongst different microparticles. Thus the microparticles,in accordance with embodiments of the present invention, allow for thegeneration of a plurality of identical reaction micro-spaces in whichfor each type of microparticle, preferably, identical or nearlyidentical analyte concentrations and/or reaction conditions areachieved. The latter scenario is of particular interest under conditionswhen the concentration of the analyte in a sample is sufficiently high.The former scenario (1 analyte bound per microparticle at a maximum) isparticularly applicable when the concentration of the analyte in asample is rather low. In one embodiment, the present invention alsorelates to the use of prefabricated microparticles as defined furtherabove, for the provision of a plurality of identical or nearly identicalreaction spaces, providing identical reaction volumes and identicalreaction conditions, e. g. for performing a detection reaction.

In one embodiment, in a collection of prefabricated microparticlesaccording to the present invention, all the microparticles are ofidentical size and thus, each of such prefabricated microparticlesprovides for and defines the same reaction volume, such reaction volumefor example serving as reaction space for a detection reaction. In oneembodiment, in a collection of prefabricated microparticles according tothe present invention, all microparticles are of the same type and arespecific for the detection of one analyte. In another embodiment, insuch collection of prefabricated microparticles according to the presentinvention, there are different types of microparticles with each typebeing specific for the detection of a different analyte. The lattercollection of microparticles according to the present invention isparticularly useful for the detection of multiple (different) analytesin one or several samples. Sometimes, as used herein, the term“prefabricated microparticle” is used herein interchangeable with theterm “digital amplification beads” (abbreviated also as “DAB”). In oneembodiment, in a collection of prefabricated microparticles according tothe present invention, such prefabricated microparticles exist asentities that are spatially totally separate from each other. In oneembodiment, in such collection of prefabricated microparticles accordingto the present invention, such collection is therefore mono-disperse. Ifnecessary, such collection of mono-disperse prefabricated microparticlesmay be provided with the prefabricated microparticles being located onor associated with a substrate. For example, such substrate may be asieve with individual wells providing just enough space to accommodate asingle prefabricated microparticle per well. Alternatively, suchsubstrate may be a substrate with regularly arranged recesses orchannels or grooves for accommodating a prefabricated microparticleeach. In one embodiment, such substrate may be a filter. In oneembodiment, the prefabricated microparticles, containing an aqueoussolution and being dispersed/suspended in a non-aqueous phase may besubjected to one or several washing steps. To this extent, they may alsobe kept on a substrate of the aforementioned kind. Alternatively and/oradditionally, the prefabricated microparticles according to embodimentsof the present invention may subsequently be exposed to a samplecontaining an analyte or suspected of containing an analyte. By virtueof a suitable capture agent being present on a prefabricatedmicroparticle, an analyte stemming from a sample surrounding themicroparticle, may be bound to the microparticle and may subsequently bedetected. The purpose of the capture agent is a local concentrating andenriching of analyte on the outside of the microparticle. The purpose ofthe detection agent is to bind the analyte or a complex between acapture agent and the analyte, and to thus make such analyte, alone orin complex with a capture agent, detectable. In one embodiment, suchdetection occurs by either detecting the detection agent which is boundto the analyte or to the complex between the capture agent and theanalyte. In another embodiment, the analyte is amplified by way of anamplification reaction, and the thus amplified product is detected bymeans of the detection agent, this being particularly preferred in thecase that the analyte is a nucleic acid and the amplification reactionis a nucleic acid amplification reaction. Examples of such nucleic acidamplification reactions are polymerase chain reaction (PCR),transcription-mediated amplification (TMA), nucleic acid sequence-basedamplification (NASBA), loop-mediated isotheinial amplification (LAMP),self-sustained sequence replication (3 SR), strand displacementamplification (SDA), rolling circle amplification (RCA), ligase chainreaction (LCR), recombinase polymerase amplification (RPA), and nickingenzyme amplification reaction (NEAR). A person skilled in the art iswell aware of any of these amplification reactions and is capable ofperforming these, as necessary. In a further embodiment, detection ofthe analyte may occur by performing first a signal amplificationreaction and subsequently detecting the thus amplified signal. In thelatter embodiment, a signal is only amplified if there is a signal inthe first place, that is, a signal only occurs when there is an analyteto be detected, and the signal amplification reaction may for example bea nucleic acid amplification if a nucleic acid is or forms part of thedetection agent. Alternatively, the signal amplification reaction may bean enzyme-based amplification of a signal, if an enzyme is or forms partof the detection agent.

In a preferred embodiment of the method of performing a digitaldetection of an analyte in a sample, a collection of prefabricatedmicroparticles according to the present invention are exposed to asample suspected of containing an analyte to be detected, and in suchstep of exposure, the number of microparticles and the number of analytemolecules in the sample are maintained or adjusted, as necessary, suchthat the binding of a single analyte molecule per microparticlepreferably follows a Poisson distribution. In a preferred embodiment ofthis method, on average, there is no more than one analyte moleculebound per microparticle. This allows the detection of a single analytemolecule per microparticle. In another embodiment, the number of analytemolecules per microparticle is maintained or adjusted, as necessary,such that, on average, in each microparticle there is an identicalnumber of analyte molecules bound per microparticle. In this latterembodiment, the microparticles thus serve to create a plurality of likedetection spaces for a detection reaction to take place.

Reference is now made to the Figures, wherein

FIG. 1 shows a schematic representation of prefabricated microparticlesin accordance with embodiments of the present invention. Panel A showsan outside view of a prefabricated microparticle (large grey circle) onwhich a number of capture agent molecules (small filled black circles)are immobilized. The prefabricated microparticle has a matrix that iscapable of taking up an aqueous solution including reaction reagents forany reaction in which the prefabricated microparticle in accordance withthe present invention is to be used. Panel B shows an embodiment of aprefabricated microparticle which has pores. Panel B shows an embodimentwherein the prefabricated microparticle according to the presentinvention is porous having a number of pores, some of which areaccessible from the outside and some of which are not. Again, thecapture agent molecules are shown as small filled black circles. Thecapture agent molecules are predominantly located on the surface of themicroparticle, such surface referring to the part of the microparticlethat is accessible from the outside of the microparticle. In accordancewith embodiments of the present invention, such twin “being accessible”is meant to refer to accessibility by or for an analyte to be detected.The prefabricated microparticles in accordance with the presentinvention may be used in detection or quantitation reactions, forexample a digital detection reaction.

FIG. 2 shows a schematic flow diagram of a detection reaction inaccordance with the present invention. To a vessel containingprefabricated microparticles according to the present invention (suchprefabricated microparticle being schematically shown in the figure as awhite circle at the bottom of the reaction vessel), there is added asample liquid including an analyte. The prefabricated microparticle inaccordance with the present invention (herein also sometimes referred toas digital amplification bead, “DAB”) after having been exposed to the(optionally labelled) analyte and after having bound the analyte by thecapture agent, is transferred to a new reaction vessel. Theprefabricated microparticles according to embodiments of the presentinvention comprise a suitable capture agent, e. g. streptavidin, that iscapable of binding the analyte of interest, in the present example thebiotinylated analyte of interest. The prefabricated microparticlesaccording to the present invention are placed on a sieve or filter ontop of the reaction vessel and washed with an appropriate wash buffer inorder to remove unbound analyte. The prefabricated microparticles inaccordance with the present invention will thus contain an aqueoussolution and, if the capture agent of the prefabricated microparticlesaccording to the present invention have bound the analyte, also thecorresponding analyte on the surface. Thereafter, the prefabricatedmicroparticles according to the present invention, resting on the sieveor filter are immersed in an oil phase, e. g. an immersion oil.Thereafter, the sieve or filter is turned upside down, and theprefabricated microparticles containing an aqueous phase including theanalyte are washed with further immersion oil to produce a suspension ofprefabricated microparticles (containing an aqueous solution includingan analyte) in an oil phase, in the present case the immersion oil usedfor washing of the prefabricated microparticles from the sieve orfilter. The microparticles in accordance with embodiments of the presentinvention also contain a detection agent allowing the detection ananalyte in a subsequent detection reaction. Such subsequent detectionreaction is then performed as a result of which certain microparticlesindicate the presence of an analyte, whereas others do not. Thosemicroparticles indicating the presence of an analyte are shown in thefigure as dark filled circles, whereas those microparticles indicating anegative result are shown as white circles.

FIG. 3 shows a similar reaction to the reaction shown in FIG. 2, but atthe level of the individual microparticle. On the left, there is shown aprefabricated microparticle in accordance with embodiments of thepresent invention, such microparticle being shown as a white circle.There are capture agent molecules located predominantly on the surfaceof the microparticle, and on the outside surrounding the microparticle,there are nucleic acids (squiggly lines) some of which are labeled witha suitable agent, e. g. biotin (square). The capture agent moleculescomprise another complementary agent, e. g. streptavidin (negative moldof a square shown on the capture agent, “pearls” decorated on thesurface of the microparticle) in this embodiment, and those nucleicacids labeled with biotin (square) will bind to the respective captureagents. It is clear that biotin and streptavidin may also be exchanged,i. e. the biotin is comprised by the capture agent, and the streptavidinis attached to the nucleic acid. Unlabeled nucleic acids will not bindand can be washed off in a subsequent wash step (see FIG. 2). Therespective microparticle containing solution is then mixed with oil andany aqueous liquid not embedded in a particle is removed from the oilphase. Thus remains a suspension of particles in an non-aqueous matrix(shown in the figure as a square frame). The material from which theprefabricated microparticle in accordance with the present invention ismade can also be liquefied in this embodiment, and if such liqueficationoccurs (see right square frame of the figure), after the microparticlehas been put into an appropriate oil phase, this will generate amicroreaction space containing an aqueous solution including an analyteto be detected and a detection reaction within an oil phase (right handsquare frame in FIG. 3).

FIG. 4 shows different colour-coded/labeled prefabricated microparticles(or digital amplification beads, “DABs”) which have been marked withdifferent dyes. Such different labels may be achieved by either choosingdifferent dye types or different dye concentrations for differentprefabricated microparticles. On the right hand panel (B), this isschematically shown by showing different microparticles according to thepresent invention which have different amounts of dye and which arespecific for different analytes. Such “encoding” may be in relation tothe specificity of the capture agent or the specificity of the detectionagent of the individual microparticle.

FIG. 5 shows examples of prefabricated microparticles in accordance withthe present invention. Panel A shows a 5×magnified transmission image ofprefabricated microparticles containing an aqueous phase in an oilphase. Panel B shows a 5×magnified transmission image of prefabricatedmicroparticles containing an aqueous phase, in phosphate buffer salinesolution (PBS). In both panels, it can be seen that the prefabricatedmicroparticles are uniform in size and provide a plurality of identicalreaction volumes/reaction spaces for a detection reaction to take place.

FIG. 6 shows a 5×magnified fluorescence image (with an excitation wavelength of 490 nm and an emission wave lengths of 510 nm) whereinprefabricated microparticles in an oil phase are shown after aPCR-amplification reaction has been performed. The prefabricatedmicroparticles were produced as described in embodiment 2 of theexamples of the present specification. As can clearly be seen there areprefabricated microparticles which have a bright fluorescence signal,and there are other prefabricated microparticles which show a low flowfluorescence signal. In those microparticles with a bright fluorescencesignal, a successful amplification of the analyte, in this case of anucleic acid, has taken place, and this is shown by the brightfluorescence signal. In other microparticles, no amplification reactionhas taken place, and this is indicated by no fluorescence or by acomparatively low(er) fluorescence signal which stems from thebackground fluorescence of the probes that have been used here (e.g.TaqMan probes).

Moreover, reference is now made to the following specific examples whichare given to illustrate, not to limit the present invention.

EXAMPLES Embodiment 1: Generation of Mono Disperse Digital AmplificationBeads (DABs)

Generation of DAB Mixes

Component 1:

Deionized Water, nuclease free

Ultra low gelling Agarose (Sigma-Aldrich, #A5030), biotin-labelled 2%(w/v)

In order to prepare biotin-labeled agarose, the Ultra-low GellingTemperature Agarose was first activated and then coupled to EZ-Link™Amine-PEG11 biotin. The activation can alternatively be carried out bybromine cyan modification, mild oxidation (generation of aldehydegroups), carbonyldiimidazole (CDI), a di- or trichlorotriazine compoundor by other methods known. Alternatively, a reactive biotin compoundsuch as, for example, a biotin-monochlorotriazine can be coupleddirectly onto agarose. Optimal biotin coverage is determined bytitration in preliminary tests in order to maximize streptavidin bindingcapacity while maintaining the matrix properties of agarose (melting andgel formation behavior, low unspecific binding).

The constituents of component 1 are pipetted together, shaken briefly ona vortex mixer and centrifuged. Subsequently, the mixture is incubatedat 65° C. at 1500 rpm in a thermoshaker in order to melt the ultra-lowgelling agarose and obtain a homogeneous agarose amplification mixture.Subsequently the component 1 mixture is cooled down to 35° C. and mixedwith an equal volume of component 2 that has been kept at the sametemperature.

Component 2 consists of 2× Platinum™ Hot Start PCR Master Mix(Invitrogen, #13000012. The DAB mixture is then kept at 35° C. untilfurther use.

Generation of Mono Disperse DABs on the Dolomite μEncapsulator System

5 mL of the emulsion reagent PicoSurf™ 5% in Novec7500 oil (DolomiteMicrofluidics) are filtered through a 0.2 μm filter, transferred to aclean 20 ml glass tube (Fisher Scientific, #12353317) and placed intothe reservoir of a pump controlling the flow of the oil phase (oil phasepump). Two other pumps controlling the flow of the agarose phase (DABmix pumps) are filled with the inert “driving liquid” HFE-7500 (DolomiteMicrofluidics, #3200425).

A “Reagent Droplet Chip” (50 μm, fluorophilic Dolomite Microfluidics,#3200445) and a “Sample Reservoir Chip” (Dolomite Microfluidics,#3200444) are placed in the μEncapsulator 1 system. The set temperatureof the Temperature control unit (TCU) is set to 35° C. A volume of 100μl of the DAB mix is added to each reservoir of the sample reservoirchip. Droplets are generated with flow rates of approx. 2 μl/min forboth DAB Mix pumps and with approx. 50 μl/min for the oil phase pump.The parameters are monitored with the Dolomite Flow Control AdvancedSoftware. The generated DABs have a volume of approx. 65 μl. Thematerial is collected in an Eppendorf tube on ice, and then stored at2-8° C.

Transfer of DABs to the Aqueous Phase and Exclusion of Non-CompliantParticles

The DABs are extracted from the oil phase by centrifugation through asieve structure. Thus beads of deviant size are removed. For thispurpose, the DAB emulsion is first applied to a tube equipped with aSEFAR PETEX® fabric w=44 μm) and centrifuged at 300×g. The oil phase andunder-sized DABs are moved through the sieve while larger DABs remain onthe SEFAR fabric. In order to completely remove the oil phase, the DABsare re-suspended in wash buffer and filtered again through the SEFARsieve. The employed wash buffer consists of 1× Taq DNA polymerase PCRbuffer [20 mM Tris HCl (pH 8.4), 50 mM KCl] (Invitrogen, #18067017) and1% TritonX100 (Sigma-Aldrich, # X100). This washing step is repeated 5times until the oil phase has been completely removed. Two additionalwashing steps are performed with 1× Taq DNA polymerase buffer withoutdetergent. DABs are recovered by applying the filter unit into asuitable centrifuge tube in opposite orientation. Wash buffer is appliedfrom the top onto the back side of the filter (the side facing away fromthe particles). The filtration unit is centrifuged for 1 min at 1.000×g.In order to recover all particles this step is repeated several times.Over-sized DABs are filtered out by pipetting the entire volume onto afilter equipped with SEFAR PETEX® tissue with a mesh width of w=59 μm(SEFAR AG, 07-59/33). The unit is briefly centrifuged at 300×g. Materialthat has passed the filter is collected and contains the DABs of thedesired size.

Coating of DABs with Streptavidin

Coating of the DABs with streptavidin is accomplished in the washingbuffer used before. The concentration of streptavidin is selected suchno accessible biotin remains on the surface of the DABs. In any caseStreptavidin is applied in excess in order to avoid cross-linking ofDABs. Optimal streptavidin concentration has been determined inpreliminary tests with labelled Streptavidin by determining a plateausurface coverage. After coupling with Streptavidin the DABs are washedseveral times on 44 μm SEFAR PETEX centrifugation units with a washbuffer without Streptavidin. Subsequently, the concentration of the DABsis determined by counting under a microscope in a DHC-N01 (NeubauerImproved) counting chamber (INCYTO) or cytometrically on the CytoFlexflow cytometer (Beckman Coulter).

The DABs are aliquoted in units containing approximately 100,000 beadsand mixed with 100 mM Trehalose. After excess buffer volume has beenremoved the DABs are lyophilized.

Embodiment 2: Application of Mono Disperse Amplification Beads (DABs)for Performing Digital PCR

Enrichment of a HIV 1 (Subtype O) Targets on DABs and Incubation ofThose Beads with a Amplification Mix

Purified HIV-1 RNA (subtype O) labeled with biotin by reversetranscription is enriched on streptavidin-modified digital amplificationbeads. The entire volume of the reverse transcription reaction is addedto a defined amount of lyophilized DABs. DABs absorb a part of theapplied liquid and swell. The beads are carefully re-suspended. In orderto avoid agglomerates ultrasound may be used. Subsequently thesuspension is applied to a centrifugation tube equipped with SEFARPETEX® tissue (w=44 μm). The supernatant is removed by centrifugation ofthe column at 300×g. For washing, the previously used wash buffer isadded without detergent to the column and also centrifuged at 300×g.Washing is repeated several times and the DABs are ultimately taken upin component 3. In this embodiment the DABs take up all the componentsnecessary for the PCR by diffusion.

Component 3 consists of the following reagents (final concentrations):

-   -   1× Platinum™ Hot Start PCR Master Mix (Invitrogen, #13000012)    -   0.2 μM sense primer: GCAGTGGCGCCCGAACAGG (Metabion international        AG)    -   0.2 μM antisense primer 1: ACTGACGCTCTCGCACCCATCT (Metabion        international AG)    -   0.2 μM antisense Primer 2: TGACGCTCTCGCACCCATCTCTC (Metabion        international AG)    -   1×SYBR® Green I nucleic acid gel stain (Sigma-Aldrich, #S9430)        or 1× EvaGreen® Fluorescent DNA stain (Jena Bioscience,        #PCR-352)

Compartmentalization by Dispersing of DABs in Oil

Micro-compartments with a defined volume are created by dispersing DABsin a fluorocarbon oil, e.g. PicoSurf™ 5% dispersed in Novec 7500 oil(Dolomite Microfluidics, #3200214. Instead of a heavy fluorocarbon oil alight mineral oil with emulsifier, e.g. Mineral oil (Sigma-Aldrich, #M5904 Sigma) with 5% (w/w) Span 80 (Sigma Aldrich, #85548) may beapplied.

The complete aqueous phase is brought in contact with an excess of oilin an Eppendorf tube. Ultrasound is applied for one minute. Both theDABs loaded with HIV-1 subtype 0 target and the supernatant of component3 are dispersed and emulsified in the oil phase. The generated aqueousdroplets of the supernatant of component 3 and the DABs differsignificantly in their volume, the droplets having a much smallervolume. The generated emulsion is pipetted onto SEFAR PETEX® tissue witha mesh width of 44 μm. Smaller droplets as well as larger droplets thatmay not contain DABs are removed by mild centrifugation. Repeatedwashing with the same oil removes all liquid droplets. By introducingthe filter unit into a suitable centrifuge tube in the oppositeorientation the concentrated DABs are extracted from the sieve. The oilwith the DABs is transferred into a detection chamber with an area ofapproximately 2 cm² and a layer thickness of approximately 1 mm. Theopposite surfaces of the chamber are made of transparent hydrophobicmaterial. If a fluorocarbon oil is used, the DABs assemble as amonolayer (dense packing) on the hydrophobic upper surface due to thedifference in density between the beads and the oil. If a mineral oil isapplied the DABs will accumulate at the lower surface. Thus the DABsprovide micro reaction containers for the subsequent digital PCR.

Amplification Reaction in DAB Micro-Compartments

DABs suspended in oil are subjected to the temperature cycling in thesame chamber on a PELTIER element 30×30×4.7 mm, 19.3 W (Quick-Ohm,Kiipper & Co. GmbH, #QC-71-1.4-3.7M). The captured cDNA is internalizedupon melting of the agarose and transformation of DABs into liquiddroplets. The amplification of individual cDNA molecules takes place inthe resulting micro-reaction compartments.

The thermal conditions applied are:

Initial denaturation for 2 min at 95° C. followed by 45 cycles ofDenaturation at 95° C. for 15 sec, Annealing at 65° C. for 15 sec andExtension at 72° C. for 30 sec. Upon completion or the thermal protocolthe content of the chamber is imaged at 21° C. in transmitted whitelight and fluorescence mode with excitation λexc=470 nm and long passemission of >496 nm. The total number of DABs and the number of thosewith a fluorescence signal above a defined intensity threshold aredetermined. The threshold value is derived from previously performedamplification reactions without template. The number of templates in thereaction is determined by applying the determined numbers of positiveand negative droplets to Poisson statistics.

Embodiment 3: Establishing Digital ELISA

Here we describe the process of establishing a digital immunoassay forthe detection of human cTnI. The assay employs immuno-PCR in a digitalformat: a DNA-labeled detection antibody and a streptavidin-labeledcapture antibody form a sandwich complex with the antigen in solution.This complex is trapped on biotin-coated agarose particles with embeddedreagents for carrying out a PCR amplification. Unbound detectionantibody, and thus the DNA label, is removed by appropriate washingsteps. The agarose particles are suspended in oil so that separatereaction compartments are formed. In the subsequent droplet PCR boundDNA-label is detected.

Detection Antibodies

The cTnI detection antibody (clone 3H9, SDIX) is labeled using theThunder-Link® PLUS Oligo Conjugation System (Innova Bioscience)according to the manufacturer's protocol and then purified. Thefollowing sequence is coupled to the antibodies:

5′GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT3′

Capture Antibody:

Clone TPC-110 (SDIX) is used as a capture antibody. This was marked byLightning link Streptavidin (Innova Bioscience) according to themanufacturer's protocol.

Preparation of DABs

Preparation of the DABs was carried out according to the methoddescribed in Example 1 with the following modification. Aftertransferring the biotin-labeled agarose particles into the aqueous phaseand eliminating unsuitable particle sizes in the exemplary embodiment,the particles are collected in 1×PCR buffer. The concentration of theparticles is adjusted to about 4.000/μl. The particles are aliquoted inunits of 25 μl.

Forming of the Immune Complex and its Capture:

Reaction Mix:

human plasma 80 μl TBS(K) pH 8.4 (20 mM Tris, 50 mM KCl pH 8.4), 10 μl0.5% TritonX-100, 10 mg/ml BS HBR-Plus (Scantibodies) 10 μl DNA labelleddetection antibody x μl Streptavidin labelled capture antibody y μl

Optimization of Antibody Concentration:

Optimal concentration of detection and capture antibodies is determinedby conventional immuno-PCR. The concentrations of the two antibodieswere systematically varied and immuno-complexes using Troponin-freeplasma (negative controls) and troponin-free plasma with defined amountsof spiked Troponin I generated. These were captured on particles, washedand subjected to conventional PCR. Optimum concentration of therespective antibodies is indicated by the lowest limit of detection andbroadest dynamic measurement range.

Generating and Capturing the Immune-Complex:

25 μl of reaction mixture (see above) is prepared with the previouslydetermined optimum concentrations of the two antibodies. The reactionmixture is incubated for 10 min at 37° C. at 800 rpm on an Eppendorfthermomixer. The mixture is subsequently mixed with an aliquot of DABparticles (100,000 particles in 25 μl 1× Taq polymerase buffer).

The mixture is incubated on a thermomixer for 5 min at 25° C. at 800rpm. During this time the binding of the streptavidin-labeled captureantibodies including the immune complexes to the DABs is accomplished.The liquid is applied to a filter with SEFAR PETEX® tissue (w=44 μm) andcentrifuged at 300×g. 5 washing steps are performed with 500 μl of TBS(K) pH 8.4 (20 mM Tris, 50 mM KCl pH 8.4), 0.05% TritonX-100, 1 mg/mlBSA. Subsequently tow additional washing steps with 1×Taq DNA polymerasePCR buffer [20 mM Tris HCl (pH 8.4), 50 mM KCl] are performed.

A PCR reaction mixture (volume 25 μl) having the following compositionis prepared:

500 nM fw-Primer (5′ AGCTCTTGATCCGGCAAACA 3′) 500 nM rev-Primer (5′GCGTCAGACCCCGTAGAAAA 3′) SYBR ® Green I nucleic acid gel stain (Sigma-Aldrich, #S9430) 1:25000 12.5 μl 2x PCR-Mastermix PCR grade Water

DABs are recovered from the sieve by placing the filter in the oppositeorientation into the tube. The PCR reaction mix is applied to thefilter. Subsequently the unit is centrifuged for 1 min at 1000×g. TheDABs are collected at the bottom of centrifuge tube and then incubatedfor 10 min in the PCR reaction mixture at 25° C. at 800 rpm on athermomixer.

Performing the PCR Reaction

DABs are transferred to the oil phase as described in embodiment 2. PCRamplification is performed over 40 cycles with the following parameters:

Cycle 1:

-   -   5 min 95° C.    -   30 sec 65° C.    -   30 sec 72° C.

Cycle 2-40:

-   -   30 sec 94° C.    -   30 sec 65° C.    -   30 sec 72° C.

After completing amplification the SYBR green signal of the individualparticles is detected by means of fluorescence microscopy. Data analysisis performed according to established algorithms for digital PCR.

Determining the Optimal Dynamic Measurement Range

Nonspecific binding of DNA-labeled detection antibody to DABs representsa critical parameter that limits the applicability of digitalimmuno-PCR. Nonspecifically bound label results in false-positive DABsafter amplification. Therefore, in digital immuno-PCR the quantificationof the analyte is achieved by determining the difference between apositive sample and a negative control.

In one extreme scenario nonspecific binding of the detection antibodycan lead to a majority of DABs with a false-positive signal in controlreactions without analytes. This is mitigated by reducing the effectiveconcentration of the detection antibody, either by gradually reducingthe concentration of the detection antibody in the assay or maintainingthe antibody concentration by increasing dilution of the DNA-labeleddetection antibody with the same antibody without DNA label.

Further modifications of the preferred embodiments are possible withoutleaving the scope of the invention, which is solely defined by theclaims./

The features of the present invention disclosed in the specification,the claims, and/or in the accompanying drawings may, both separately andin any combination thereof, be material for realizing the invention invarious forms thereof.

1-21. (canceled)
 22. A method of performing a detection of an analyte ina sample, said method comprising the steps: a) providing a prefabricatedmicroparticle which has a surface and includes a void volume forreceiving an aqueous solution, wherein said particle is dispersible in anon-aqueous medium and, upon dispersion in a non-aqueous medium,provides for a defined reaction space in such non-aqueous medium, inwhich defined reaction space a chemical or biochemical reactionindicating the presence of an analyte can be performed, and wherein saidprefabricated microparticle comprises a capture agent that, uponexposure of said microparticle to a sample surrounding saidprefabricated microparticle and containing an analyte, selectively andspecifically binds the analyte to be detected and that, upon binding ofthe analyte to the capture agent, forms a complex between said captureagent and said analyte, wherein said capture agent binds the analytefrom a sample surrounding said prefabricated microparticle, and whereinsaid prefabricated microparticle further comprises a detection agentthat is specific for the analyte or said complex between said captureagent and said analyte, and that binds said analyte or said complexbetween said capture agent and said analyte; b) exposing saidprefabricated microparticle to a an aqueous sample suspected ofcontaining an analyte to be detected, thus allowing the capture agent toselectively and specifically bind the analyte to be detected, ifpresent; c) placing the prefabricated microparticle into a non-aqueousphase, e.g. an oil phase and using the void volume of said prefabricatedmicroparticle as a defined reaction space in which a chemical orbiochemical reaction indicating the presence of an analyte, isperformed, by either d1) detecting the detection agent bound to saidanalyte or to said complex between said capture agent and said analyte;or d2) amplifying the analyte, if present, by way of an amplificationreaction, and detecting the thus amplified product by means of saiddetection agent, wherein said analyte is a nucleic acid and saidamplification reaction is a nucleic acid amplification, or d3)performing a signal amplification reaction and detecting the thusamplified signal, wherein said reaction space in which said chemical orbiochemical reaction indicating the presence of an analyte, isperformed, is defined by said void volume of said prefabricatedmicroparticle and is not larger than said void volume of saidprefabricated microparticle.
 23. The method according to claim 22,wherein said prefabricated microparticle is provided as a prefabricatedmicroparticle which is dried.
 24. The method according to claim 22,wherein said prefabricated microparticle is not a microparticle that isin-situ generated at the site or in the reaction, at or during whichanalyte detection is to take place.
 25. The method according to claim22, wherein the capture agent is predominantly located on the surface ofsaid prefabricated microparticle, such that the prefabricatedmicroparticle is capable of enriching and concentrating an analytelocated outside of the microparticle.
 26. The method according to claim23, wherein said prefabricated microparticle is reconstituted in anaqueous solution either during step a) or step b), and, uponreconstitution, receives such aqueous solution in its void volume. 27.The method according to claim 22, wherein said detection agent isincluded in said prefabricated microparticle during a prefabricationprocess or is included in an aqueous solution resulting fromreconstitution of the microparticle either during step a) or step b),and thus becomes part of the prefabricated microparticle uponreconstitution.
 28. The method according to claim 22, wherein saidprefabricated microparticle is made of a gel-forming agent, suchgel-forming agent being liquefiable upon the application of heat orlight, or upon a change of pH, redox potential, ionic strength,temperature, magnetic field or electromagnetic radiation, or uponexposure to an enzyme or, if the gel-forming agent itself comprises anenzyme, to a substrate of such enzyme, or any combination of theforegoing.
 29. The method according to claim 28, wherein saidgel-forming agent forms a matrix defining the surface and the voidvolume of said microparticle.
 30. The method according to claim 28,wherein said gel-forming agent is selected from a) synthetic polymersprepared from their corresponding monomers; b) silicone-based polymers;and c) naturally occurring polymers selected from polysaccharides,polypeptides, polynucleotides, and combinations thereof.
 31. The methodaccording to claim 22, wherein said capture agent is selected fromantibodies or antibody fragments, nucleic acids, Spiegelmers, andnon-antibody proteins capable of specifically binding an analyte oranalyte complex.
 32. The method according to claim 22, wherein saiddetection agent is selected from antibodies or antibody fragments,nucleic acids, Spiegelmers, and non-antibody proteins, each of themoptionally being labelled with a suitable reporter molecule thatproduces an optically or otherwise detectable signal indicating thepresence of the analyte to be detected.
 33. The method according toclaim 22, wherein said prefabricated microparticle is specificallylabelled.
 34. The method according to claim 22, which is performed usinga collection of prefabricated microparticles.
 35. The method accordingto claim 34, wherein, in said collection of prefabricatedmicroparticles, said prefabricated microparticles are different fromeach other in that they are specific for different analytes to bedetected, wherein each prefabricated microparticle is specificallylabelled such that different prefabricated microparticles and theircorresponding detected analytes can be distinguished by the specificlabels of the prefabricated microparticles.
 36. The method according toclaim 22, wherein said method involves the use of a prefabricatedmicroparticle or of a collection of prefabricated microparticles, forperforming a digital detection of an analyte or a plurality of analytesin a sample or for enriching and concentrating a plurality of analytesin a plurality of defined volumes, wherein all of said defined volumesin said plurality of defined volumes are equal.
 37. The method accordingto claim 22, wherein, after the step of exposing b), there is one orseveral washing steps.
 38. The method according to claim 22, wherein instep a), said prefabricated microparticles are provided in dried form,and, in step b), said prefabricated microparticles are reconstituted inaqueous solution and then exposed to a sample suspected of containing ananalyte to be detected, wherein, optionally after the step ofreconstituting, there is one or several washing steps.
 39. The methodaccording to claim 22, wherein, in step b) the number of prefabricatedmicroparticles and the number of analyte molecules in the sample aremaintained or adjusted, as necessary, such that the binding of a singleanalyte molecule per prefabricated microparticle follows a Poissondistribution such that, on average, there is no more than one analytemolecule bound per microparticle, thus allowing the detection of asingle analyte molecule per prefabricated microparticle.
 40. The methodaccording to claim 22, wherein, during step c), the prefabricatedmicroparticle or the collection of prefabricated microparticles issuspended in the non-aqueous phase and/or is located on a solidsubstrate isolating each prefabricated microparticle from otherprefabricated microparticles, if present, wherein said solid substrateis a filter, a sieve, a substrate having a pattern of wells, recesses,grooves, channels, trenches, craters, holes, pillars or any combinationof the foregoing.
 41. The method according to claim 22, wherein, duringor after step c), the gel-forming agent is liquefied through theapplication of heat or light, or by a change of pH, redox potential,ionic strength, temperature, magnetic field or electromagneticradiation, or upon exposure to an enzyme or, if the gel-forming agentitself comprises an enzyme, to a substrate of such enzyme, or anycombination of the foregoing, resulting in an aqueous droplet in anon-aqueous phase.